Organic compounds

ABSTRACT

The present invention relates to isolated polypeptides which comprise an amino acid sequence consisting of a mutated functional Abl kinase domain, said mutated functional kinase domain being resistant to inhibition of its tyrosine kinase activity by N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1 -ylmethyl)-benzamide or a salt thereof, to the use of such polypeptides to screen for compounds which inhibit the tyrosine kinase activity of such polypeptides, to nucleic acid molecules encoding such polypeptides, to recombinant vectors and host cells comprising such nucleic acid molecules and to the use of such nucleic acid molecules in the production of such polypeptides for use in screening for compounds which inhibit the tyrosine kinase activity of such polypeptides.

This application is a Continuation Application of Ser. No. 11/343,891,filed Jan. 31, 2006, which is a Continuation Application of Ser. No.10/263,480, filed Oct. 3, 2002, which claims benefit of ProvisionalApplication No. 60/327,387, filed Oct. 5, 2001.

FIELD OF THE INVENTION

This invention relates to isolated polypeptides which comprise an aminoacid sequence consisting of a mutated functional Abl kinase domain, saidmutated functional kinase domain being resistant to inhibition of itstyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof, to the use of such polypeptides to screen forcompounds which inhibit the tyrosine kinase activity of suchpolypeptides, to nucleic acid molecules encoding such polypeptides, torecombinant vectors and host cells comprising such nucleic acidmolecules and to the use of such nucleic acid molecules in theproduction of such polypeptides for use in screening for compounds whichinhibit the tyrosine kinase activity of such polypeptides.

BACKGROUND OF THE INVENTION

Bcr-Abl, a constitutively activated tyrosine kinase resulting from theformation of the Philadelphia chromosome [Nowell P. C. and Hungerford D.A., Science 132, 1497 (1960)] by reciprocal translocation between thelong arms of chromosomes 9 and 22 [Rowley J. D., Nature 243, 290-293(1973)], has been established as the characteristic molecularabnormality present in virtually all cases of chronic myeloid leukemia(CML) and up to 20 percent of adult acute lymphoblastic leukemia (ALL)[Faderl S. et al., N Engl J Med 341, 164-172 (1999); Sawyers C. L., NEngl J Med 340, 1330-1340 (1999)]. Bcr-Abl is sufficient to cause CML inmice [Daley G. Q. et al., Science 247, 824-830 (1990)] and itstransforming capacity is absolutely dependent on tyrosine kinaseactivity [Lugo T. G. et al., Science 247, 1079 (1990)]. The compoundN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamide(hereinafter also referred to as “STI571”; STI571 is described in EP 0564 409 and, in the form of the methane sulfonate salt, in WO 99/03854),a competitive inhibitor at the ATP-binding site of Bcr-Abl, as well asof the receptor for platelet-derived growth factor, and c-kit tyrosinekinase [Lugo T. G. et al., Science 247, 1079 (1990)], has been shown tobe capable of very rapidly reversing the clinical and hematologicalabnormalities of CML in chronic phase and in blast crisis as well as ofPh-chromosome-positive (Ph+) acute lymphoblastic leukemia (Ph+ ALL)[Druker B. J. et al., N Engl J Med 344, 1031-1037 (2001); Druker B. J.et al., N Engl J Med 344, 1038-1042 (2001)]. Whereas almost all chronicphase CML patients durably respond, remissions in CML blast crisis andPh+ ALL are transient, and most patients relapse after several months,despite continued therapy with STI571 [Druker B. J. et al., N Engl J Med344, 1038-1042 (2001)]. The mechanism of resistance to STI571 is subjectof intense research.

It was now surprisingly found that mutations present in the kinasedomain of the Bcr-Abl gene of patients suffering from CML or Ph+ ALLaccount for the biological resistance of these patients towards STI571treatment in that said mutations lead to resistance of the Bcr-Abltyrosine kinase towards inhibition by STI571.

These findings are extremely valuable in e.g. finding new compounds orcombinations of compounds which are capable to overcome resistancetowards treatment with STI571. Moreover, knowledge of such mutations isalso very useful in the diagnosis of Ph+ leukemias in that it allowse.g. the detection of drug-resistant clones before clinical relapse ofthe patient.

DEFINITIONS

Within the context of this disclosure the following expressions, termsand abbreviations have the meanings as defined below:

In the expression “a mutated functional Abl kinase domain”, the part“mutated Abl kinase domain” refers to the native human Abl kinase domaincontaining mutations including amino acid exchanges, amino aciddeletions and/or amino acid additions.

In the expression “a mutated functional Abl kinase domain”, the term“functional” indicates that the respective kinase domain possessestyrosine kinase activity. Preferably, the kinase activity of the mutatedfunctional Abl kinase domain is in the range of that of the native humanAbl kinase domain.

In the expression “a mutated functional Abl kinase domain beingresistant to inhibition of its tyrosine kinase activity by STI571 or asalt thereof”, the term “resistant” means that STI571 inhibits therespective mutated functional Abl kinase domain with an IC₅₀ that ishigher than that of the native human Abl kinase domain, i.e. higher thanabout 0.025 μM, preferably higher than about 0.15 μM, more preferablyhigher than about 0.25 μM, most preferably higher than about 5 μM.

In the expression “amino acid sequence of the native human Abl kinasedomain or an essentially similar sequence thereof, the part “or anessentially similar sequence thereof” refers to the amino acid sequenceof the native human Abl kinase domain containing mutations, includingamino acid exchanges, amino acid deletions and/or amino acid additions,that are not essential for the functionality of the kinase and itsresistance to inhibition by STI571 or a salt thereof within the meaningof the term “functional” and “resistant” as defined hereinabove.

The expression “replaced by another amino acid” refers to thereplacement of a certain natural amino acid by another natural aminoacid.

The names of the amino acids are either written out or the one letter orthree letter codes are used. Mutations are referred to by acceptednomenclature, e.g. “Ala380Thr” or “380 Ala→Thr” both indicating thatalanine at position 380 is replaced by threonine.

SEQ ID NO:1 represents the cDNA coding for the native human Abl protein(human c-abl mRNA; GenBank Accession No.: X16416).

SEQ ID NO:2 represents the amino acid sequence of the native human Ablprotein (human c-Abl; SwissProt Acc. No.: P00519).

Unless indicated otherwise, the number given for a certain amino acidrefers to the numbering of the amino acids in SEQ ID NO:2. In an aminoacid sequence that is essentially similar to the amino acid sequence ofthe native human Abl kinase domain within the meaning as defined above,the amino acids are numbered in accordance with the numbering of theamino acids in SEQ ID NO:2.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring).

A “host cell”, refers to a prokaryotic or eukaryotic cell that containsheterologous DNA that has been introduced into the cell by any means,e.g., electroporation, calcium phosphate precipitation, microinjection,transformation, viral infection, and the like.

DESCRIPTION OF THE INVENTION

In practicing the present invention, many conventional techniques inmolecular biology, microbiology, and recombinant DNA are used. Thesetechniques are well known and are explained in, for example, CurrentProtocols in Molecular Biology, Volumes I, II, and III, 1997 (F. M.Ausubel ed.); Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II, 1985(D. N. Glover ed.); Oligonucleotide Synthesis, 1984 (M. L. Gait ed.);Nucleic Acid Hybridization, 1985, (Hames and Higgins); Transcription andTranslation, 1984 (Hames and Higgins eds.); Animal Cell Culture, 1986(R. I. Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL Press);Perbal, 1984, A Practical Guide to Molecular Cloning; the series,Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors forMammalian Cells, 1987 (J. H. Miller and M. P. Calos eds., Cold SpringHarbor Laboratory); and Methods in Enzymology Vol. 154 and Vol. 155 (Wuand Grossman, and Wu, eds., respectively).

In particular, the polypeptides of the present invention can be producedby recombinant DNA technology using techniques well-known in the art.Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing the sequences encoding thepolypeptides of the invention and appropriatetranscriptional/translational control signals. A variety ofhost-expression vector systems can be utilized to express thepolypeptides of the invention.

(1) The invention relates to an isolated polypeptide which comprises amutated functional Abl kinase domain that is resistant to inhibition ofits tyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

(2) The invention further relates in particular to an isolatedpolypeptide which comprises a mutated functional Abl kinase domaincomprising the amino acid sequence of the native human Abl kinase domainor an essentially similar sequence thereof in which at least one aminoacid is replaced by another amino acid, said mutated functional Ablkinase domain being resistant to inhibition of its tyrosine kinaseactivity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

(3) The invention especially relates to an isolated polypeptide whichcomprises a mutated functional Abl kinase domain comprising the aminoacid sequence of the native human Abl kinase domain or an essentiallysimilar sequence thereof in which at least one amino acid selected fromLeu248, Glu255, Lys271, Glu286, Met290, Thr315, Tyr320, Asn322, Glu373,His375 and Ala380 is replaced by another amino acid, said mutatedfunctional Abl kinase domain being resistant to inhibition of itstyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

(4) A preferred embodiment of the invention relates to an isolatedpolypeptide which comprises a mutated functional Abl kinase domaincomprising the amino acid sequence of the native human Abl kinase domainor an essentially similar sequence thereof in which at least one aminoacid selected from Leu248, Glu255, Lys271, Glu286, Met290, Tyr320,Asn322, Glu373, His375 and Ala380 is replaced by another amino acid,said mutated functional Abl kinase domain being resistant to inhibitionof its tyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

(5) Another preferred embodiment of the invention relates to an isolatedpolypeptide which comprises a mutated functional Abl kinase domaincomprising the amino acid sequence of the native human Abl kinase domainor an essentially similar sequence thereof in which at least one aminoacid selected from Leu248, Lys271, Glu286, Met290, Tyr320, Asn322,Glu373, His375 and Ala380 is replaced by another amino acid, saidmutated functional Abl kinase domain being resistant to inhibition ofits tyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

(6) Another especially preferred embodiment of the invention relates toan isolated polypeptide which comprises a mutated functional Abl kinasedomain comprising the amino acid sequence of the native human Abl kinasedomain or an essentially similar sequence thereof in which at least oneamino acid selected from Glu255, Thr315 and Ala380 is replaced byanother amino acid, said mutated functional Abl kinase domain beingresistant to inhibition of its tyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

(7) Another very preferred embodiment of the invention relates to anisolated polypeptide which comprises a mutated functional Abl kinasedomain comprising the amino acid sequence of the native human Abl kinasedomain or an essentially similar sequence thereof in which at least oneamino acid selected from Glu255 and Ala380 is replaced by another aminoacid, said mutated functional Abl kinase domain being resistant toinhibition of its tyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

(8) Most preferably the invention relates to an isolated polypeptideaccording to any one of the preceding paragraphs (2)-(7), wherein in theamino acid sequence of the native human Abl kinase domain or anessentially similar sequence thereof a single amino acid is replaced byanother amino acid.

(9) The invention relates very especially preferred to an isolatedpolypeptide which comprises a mutated functional Abl kinase domaincomprising the amino acid sequence of the native human Abl kinase domainor an essentially similar sequence thereof in which Glu255 is replacedby another amino acid, said mutated functional Abl kinase domain beingresistant to inhibition of its tyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

(10) Most especially preferred the invention relates to an isolatedpolypeptide which comprises a mutated functional Abl kinase domaincomprising the amino acid sequence of the native human Abl kinase domainor an essentially similar sequence thereof that contains at least oneamino acid mutation selected from Glu255Val, Glu255Lys, Thr315Val,Thr315Leu, Thr315Met, Thr315Gln, Thr315Phe and Ala380Thr, said mutatedfunctional Abl kinase domain being resistant to inhibition of itstyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

(11) In a further very preferred embodiment the invention relates to anisolated polypeptide which comprises a mutated functional Abl kinasedomain comprising the amino acid sequence of the native human Abl kinasedomain or an essentially similar sequence thereof that contains at leastone amino acid mutation selected from Glu255Val, Thr315Val, Thr315Leu,Thr315Met, Thr315Gln, Thr315Phe and Ala380Thr, said mutated functionalAbl kinase domain being resistant to inhibition of its tyrosine kinaseactivity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

(12) In another especially preferred embodiment the invention relates toan isolated polypeptide which comprises a mutated functional Abl kinasedomain comprising the amino acid sequence of the native human Abl kinasedomain or an essentially similar sequence thereof that contains at leastone amino acid mutation selected from Thr315Leu, Thr315Met, Thr315Glnand Thr315Phe, said mutated functional Abl kinase domain being resistantto inhibition of its tyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

(13) Most preferably the invention relates to an isolated polypeptideaccording to any one of the preceding paragraphs (10)-(12), wherein theamino acid sequence of the native human Abl kinase domain or anessentially similar sequence thereof contains a single amino acidmutation.

(14) Preferred above all the invention relates to an isolatedpolypeptide which comprises a mutated functional Abl kinase domaincomprising the amino acid sequence of the native human Abl kinase domainor an essentially similar sequence thereof that contains the amino acidmutation Glu255Val, said mutated functional Abl kinase domain beingresistant to inhibition of its tyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

(15) In a preferred embodiment the invention relates to an isolatedpolypeptide according to any one of the preceding paragraphs (2)-(14),wherein the amino acid sequence of the native human Abl kinase domainconsists of amino acids 229-500 of SEQ ID NO:2.

(16) In another preferred embodiment the invention relates to anisolated polypeptide according to any one of the preceding paragraphs(2)-(15), said isolated polypeptide being a Bcr-Abl tyrosine kinase.

(17) In yet another preferred embodiment the invention relates to theuse of an isolated polypeptide of any one of the preceding paragraphs(2) to (16) to screen for compounds which inhibit the tyrosine kinaseactivity of said polypeptide.

(18) The invention also relates to an isolated nucleic acid moleculecomprising a nucleotide sequence that encodes a polypeptide according toany one of the preceding paragraphs (2)-(16).

(19) The invention further relates to the use of a nucleic acid moleculeof the preceding paragraph (18) in the production of a polypeptide ofany one of the preceding paragraphs (2) to (16) for use in screening forcompounds which inhibit the tyrosine kinase activity of saidpolypeptide.

(20) The invention also relates to a recombinant vector comprising anucleic acid molecule according to the preceding paragraph (18).

(21) The invention further relates especially to a recombinant vectoraccording to the preceding paragraph (20), which is a recombinantexpression vector.

(22) The invention also relates to a host cell comprising a recombinantvector according to the preceding paragraph (20) or (21).

Preferably the invention relates to an isolated polypeptide whichcomprises a mutated functional Abl kinase domain comprising the aminoacid sequence of the native human Abl kinase domain in which at leastone amino acid is replaced by another amino acid, said mutatedfunctional Abl kinase domain being resistant to inhibition of itstyrosine kinase activity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.

Most preferred are the mutations described herein, which are present inpatients who suffer from Philadelphia chromosome-positive leukemia andare resistant against treatment with STI571.

A preferred salt of STI571 is the methane sulfonate salt described in WO99/03854.

Screening for compounds which inhibit the tyrosine kinase activity ofthe polypeptides of the invention may be done for example by using anisolated polypeptide of the invention in any in vitro tyrosine kinasephosphorylation assay known in the art and determining the potential ofa compound to inhibit the tyrosine kinase activity of a polypeptide ofthe invention in such an assay.

High-throughput screening assays known in the art may be used to screenlarge compound libraries for compounds which inhibit the tyrosine kinaseactivity of the polypeptides of the invention.

Besides the random screening of large compound libraries, thepolypeptides of the present invention may also be used in the followingscreening approach: The 3-dimensional structure of a polypeptide of theinvention is determined by e.g. X-ray crystallography. The atomiccoordinates of a polypeptide of the invention are then used to design apotential inhibitor. Said potential inhibitor is then synthesized andtested for its ability to inhibit the tyrosine kinase activity of thepolypeptide of the invention in any in vitro tyrosine kinasephosphorylation assay.

EXAMPLES

The following Examples serve to illustrate the invention withoutlimiting its scope.

Example 1 Methods Plasmids and Site Directed Mutagenesis:

The hybrid cDNA coding for HckAblSH1 was cloned by amplifying therespective DNA fragments from pUCΔNdel/XbalHck [Warmuth M. et al., J.Biol. Chem. 272, 33260-70 (1997)] and pcDNA3bcr-abl. These fragmentswere ligated blunt end to yield pUCΔNdel/XbalhckablSH1. Because of itsrelatively small size when compared to Bcr-Abl or c-Abl, this construct,hckAblSH1, allowed to introduce point mutations into the kinase domainof Abl by a one step cloning procedure. Point mutations were introducedinto hckablSH1 using the QuickChange site directed mutagenesis protocolfrom Stratagene (La Jolla, Calif.). In order to introduce pointmutations into Bcr-Abl, a Kpnl/Eco 47III-subfragment of Bcr-Ablcontaining the sequence coding for Bcr-Abl's kinase domain was clonedinto pUCΔNdel/Xbal engineered by site directed mutagenesis to contain anEco47III site in the polylinker. After introduction of point mutations,this fragment was first recloned into pcDNA3abl. Thereafter, the 5′ partof abl up to the Kpnl site was substituted by Bcr coding sequences usinga Kpnl-fragment from pcDNA3bcr-abl. All mutations were confirmed bysequencing. For expression in Cos7 and 32D cells cDNAs were cloned intopApuro.

Cell Lines:

Parental 32D cells as well as 32D cells expressing Bcr-Abl and mutantsthereof (32Dp21) were grown in Iscove's modified dulbeccos media (IMDM)supplemented with 10% FBS. COS7 cells were cultured in Dulbecco'smodified eagle medium (DMEM) containing 4,5 g/l glucose) andsupplemented with 10% FBS. All media and FBS were purchased from GibcoLife Technologies, Inc, Karlsruhe, Germany.

Transfection of Cells:

Cos7 cells were transfected using Effecten® transfection reagent as tothe guidelines of the manufacturer (Quiagen, Hilden, Germany). 32D cellswere transfected by electroporation. Puromycin was used for selection ata concentration of 1 μg/ml.

Cell Lysis:

Cos7 cells were lysed as described recently [Warmuth M. et al., J. Biol.Chem. 272, 33260-70 (1997)]. For lysis, exponentially growing 32D cellswere harvested and washed twice in cold PBS. For experiments evaluatingthe activity profile of STI571, cells were incubated with the indicatedconcentrations of inhibitor or with DMSO at a density of 5×10⁶ cells/mlfor 1.5-2h. 10⁷ cells were lysed in 100 μl of lysis buffer containing 1%NP-40, 20 mM Tris(pH 8,0), 50 mM NaCl, and 10 mM EDTA, 1 mMphenylmethylsulfonylfluorid, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and2 mM sodium orthovanadate. After resuspension in lysis buffer, cellswere incubated for 25 min on ice. Finally, unsoluble material wasremoved by centrifugation at 15,000 g. Clarified lysates were checkedfor protein concentrations using a BioRad protein assay.

Immunoprecipitation:

For immunoprecipitation 150 μl of standardized 32D cell lysate wasdiluted by addition of 450 μl of incubation buffer containing 20 mMTris(pH 8,0), 50 mM NaCl, and 10 mM EDTA, 1 mMphenylmethylsulfonylfluorid, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and2 mM sodium orthovanadate and incubated with 5 μg of the indicatedantibodies for 2 hours on a overhead rotor at 4° C. Sepharose A beads(Pharmacia Biotech Inc., Freiburg, Germany) were prepared by washingtwice in IP buffer [0,1% NP-40, 20 mM Tris(pH 8,0), 50 mM NaCl, and 10mM EDTA, 1 mM phenylmethylsulfonylfluorid, 10 μg/ml aprotinin, 10 μg/mlleupeptin, and 2 mM sodium orthovanadate] and added to each sample for 2additional hours. Finally, the immunoprepicitates were washed threetimes in IP buffer, subsequently boiled in 2× sample buffer and preparedfor SDS-PAGE.

Gel Electrophoresis and Immunoblotting:

Gel electrophoresis and immunoblotting were performed as described[Danhauser-Riedl S. et al., Cancer Res. 56, 3589-96 (1996); Warmuth M.et al., J. Biol. Chem. 272, 33260-70 (1997)] with some minormodifications. Proteins were routinely blotted on nitrocellulosemembranes (Schleicher & Schuell, Dassel, Germany). Membranes wereblocked in 5% milk powder for 1 h. Primary and secondary antibodies werediluted 1:500 to 1:5000 in TBS containing 1% milk powder. Proteins weredetected using the ECL or ECL Plus detection system as recommended bythe manufacturer (Amersham, Braunschweig, Germany).

Detection of Apoptosis by Flow Cytometry:

For assessing apoptosis induced by the various kinase inhibitors, cellswere incubated with the indicated concentrations of STI571 at a densityof 5×10⁴ per ml. Apoptosis was routinely assessed by measuring thebinding of FITC-conjugated Annexin V to the membranes of apoptosingcells. About 5×10⁴ cells were taken at the indicated time points andwashed once in PBS. Thereafter, cells were resuspended in 195 μl ofAnnexin V binding buffer and 5 μl of Annexin V-FITC (Bender MedSystemsDiagnostics, Vienna, Austria) were added. Cells were mixed and incubatedat room temperature for 10-20 min. Afterwards, cells were pelletedagain, washed once and resuspended in 190 μl of Annexin V bindingbuffer. 10 μl of a 20 μg/ml propidium iodide stock solution were addedand the ratio of Annexin V-positive to negative cells was determined byFACS-analysis using a Coulter EPICS XL 4-color cytometer.

Results:

Mutations to either Valine (V), Leucine (L), Isoleucine (I), Methionine(M), Glutamine (Q) or Phenylalanine (F) at position 315 and to eitherSerine (S), Cysteine (C) or Threonine (T) at position 380 were generatedin a hybrid kinase, HckAblSH1, consisting of the SH2 and SH3 domain ofHck and the SH1/kinase domain of Abl. When expressed in Cos7 cells,these hybrid kinases and all mutants at position 315 and 380 showed ahigh spontaneous kinase activity, proving that these positions are notcritical for ATP binding (Table 1). In marked contrast, when tested forinhibition by STI571, no inhibition was seen with up to 10 μM ofcompound for the mutants T315L, T315I, T315M, T315Q and T315F (Table 1),whereas HckAblSH1 wild-type (wt) could be inhibited with similarkinetics by STI571 as were found for Bcr-Abl (IC50 cellular tyrosinephosphorylation (IC50_(CTP)) approx. 0.5 μM). The mutants T315V andA380T retained some partial sensitivity but IC50_(CTP) values were stillhigher than 10 μM. In contrast, the mutants A380S and A380C displayedsensitivity to STI571, which was comparable to HckAblSH1 wt (see Table 1for summary).

TABLE 1 Influence of mutations of T315 and A380 of HckAblSH1 on kinaseactivity and inhibiton by STI571 kinase activity Inhibition by STI571T315V +++ IC50 > 10 T315L +++ CR T315I +++ CR T315M ++++ CR T315Q ++ CRT315F ++ CR A380C +++ NS A380S +++ NS A380T + IC50 > 10 All data basedon inhibiton of cellular tyrosine phosphorylation of transientlytransfected Cos7 cells determined by Western blot analysis using themonoclonal α-phosphotyrosine antibody PY99. IC50 values were determinedusing scion image software. Complete remission (CR) was defined as nodetectable reduction of cellular tyrosine phosphrylation by 10 μMSTI571. NS (normal sensitivity) = inhibtion with similar kinetics asHckAblSH1 wt.

Our data identify positions 315 and 380 as critical gatekeepers for thebinding pocket of STI571, which contribute to define the sensitivity ofindividual protein kinases towards STI571. For example, theSTI571-insensitive receptor tyrosine kinase Flt-3, which has highhomology to the c-Kit and the PDGF-R kinases, has a phenylalanine at theposition homologous to T315, which would, based on our data, not be inaccordance with STI571 binding. In a similar way, the resistance of mostother kinases tested with STI571 could be explained.

In order to investigate whether and to what degree some of the abovedescribed point mutations of the gatekeeper position T315 are able toinduce biological resistance towards STI571 we introduced into fulllength Bcr-Abl the mutations T315V, T315L, T3151, T315M, T315Q andT315F. When expressed in Cos7 cells all mutants displayed kinaseactivity close to or similar to wild-type Bcr-Abl (Bcr-ABLwt). If testedfor inhibition by STI571, identical results were obtained as describedfor the corresponding mutations in HckAblSH1 (Table 2). Similar toBcr-Ablwt, expression of these mutants in 32D, an IL-3-dependent,hematopoietic cell line of murine origin, gave rise to cell linesgrowing IL-3 independently. Exposure of 32 DBcr-Ablwt cells to 1 or 10μM STI571 lead to a rapid stop of proliferation and induction ofapoptotic cell death in more than 90% of cells. On the contrary, if T315mutant Bcr-Abl kinases, for example T315, were expressed the block inproliferation and the induction of apoptosis caused by 1 μM STI571 werecompletely abolished (Table 2) and the effects of STI571 seen at 10 μMwere reduced to levels found in control experiments using parental 32Dcells grown in the presence of IL-3. Phosphotyrosine blots of samples ofcells expressing either wt or mutant Bcr-Abl proteins confirmed thatmutations at position 315 completely abolished the effect of STI571 onAbl auto- and substrate phosphorylation, with the exception of T315Vwhich was still to some degree inhibited by STI571 but displayed asimilar biological phenotype as the other mutants (Table 2). Thissuggests that the reminder biological activity of STI571 at 10 μM wasrather due to cytotoxicity than to a reminder sensitivity of the mutantsor cross-reaction of STI571 with another tyrosine kinase. In summary,all mutations lifted the IC50 for inhibition of proliferation(IC50_(IOP)) from 0.09 to approximately 7.5 μM and for inhibition ofsurvival (IC50_(IOS)) from 0.5 to more than 10 μM (Table 2). Takentogether, these data show that mutations of “molecular gatekeeper”positions as described above are able to confer complete biologicalresistance towards STI571 in a cell culture model.

TABLE 2 Biochemical and biological characterization of mutations of T315in Bcr-Abl to amino acids with longer side chains IC50 (μM) InductionIC50 cellular of factor- (μM) tyrosine independent IC50 (μM) prolif-kinase phosphorylation growth apoptosis eration mutant activity Cos7 32Din 32D 32D 32D (32D) <10 7.5 wt +++ 0.25 0.25 yes 0.5 0.09 T315V+++ >10 >10 yes >10 7.5 T315L ++++ c.r. c.r. yes >10 7.5 T315I +++ c.r.c.r. yes >10 7.5 T315M ++++ c.r. c.r. yes >10 7.5 T315Q +++ c.r. c.r.yes >10 7.5 T315F +++ c.r. c.r. yes >10 7.5 c.r.: complete remission (nodetectable reduction of cellular tyrosine phosphrylation by 10 μMSTI571)

Example 2

STI571 inhibits the Abl tyrosine kinase with an IC₅₀ of 0.025 μM forpurified Bcr-Abl and c-Abl but not the fms or the Src family kinases.The mechanism of inhibition is through competitive inhibition of ATPbinding. To better understand the mechanism of specificity of thetyrosine kinase inhibitor the Abl kinase was compared to a model of theLck kinase domain. This model predicts the following sites are criticalfor STI571 association: L248, Y320, N322, E373, H375 and A380. Each ofthese residues were changed to the corresponding residue in Src or fmsand IC₅₀ values for STI571 with each mutant were determined. L248A andH375L yielded kinase inactive mutants, Y320K, N322S, E373N and A380G hadIC₅₀ values identical to wild type Abl. A380T, however, demonstrated anIC₅₀ of 0.34 μM suggesting that STI571 bound less efficiently when alarger residue replaced the alanine. Recent crystallization of the Ablkinase domain with a related inhibitor shows that the configuration ofthe activation loop of the Abl kinase domain differs significantly fromthat of the Src family kinases. This structure identifies K271, E286,M290, T315, M318 and D381 as critical contacts of STI571. All of theseresidues are conserved between Src and Abl. The last two of these bindSTI571 via their peptide backbone, thus mutants in these residues cannotbe created. The remainder of the residues were mutated to residueslacking the potential for hydrogen bonding and IC₅₀ values weredetermined. K271R, E286L and M290A were kinase inactive. T315V had anIC₅₀ value of 0.35 μM, which is consistent with the crystal structure ofthe Abl kinase domain which predicts that the side chain of T315 forms acritical hydrogen bond with STI571.

Example 3

A group of 32 patients who are either refractory to treatment withSTI571 or who relapsed whilst being treated were investigated. Themedian duration of therapy was 95 days; prior to STI571 treatment, twopatients were in chronic phase, nine in accelerated phase, 20 in myeloidand, and one in lymphoid blast crisis of the disease. Reversetranscriptase-polymerase chain reaction (RT-PCR) products specific forthe Bcr-Abl tyrosine kinase domain were sequenced (Heminested RT-PCR wasperformed to amplify the sequence specifically coding for the Bcr-Abltyrosine kinase: 1^(st) step B2B ACAGAATTCCGCTGACCATCAATAAG andA7−AGACGTCGGACTTGATGGAGAACT; 2^(nd) step FA4+ AAGCGCAACAAGCCCACTGTCTATand A7−).

An acquired A→T point mutation at position 58802 (GeneBank accessionnumber U07563, locus HSABLGR3)—which results in a Glu255Valsubstitution—was detected in one patient. Restriction analysis of cDNAand genomic DNA (RT-PCR and genomic PCR were performed using primers A4+TCACCACGCTCCATTATCCA, A4− CTTCCACACGCTCTCGTACA; Mnl I restriction digestof PCR products; removal of an Mnl I restriction site as the result ofthe point mutation A58802T) was used to confirm the presence of themutation and to track it during the course of treatment. Only wild-typeAbl sequence was present before the STI571 therapy. The patient wastreated with STI571 in late chronic phase, went into completehematologic remission, but progressed to blast crisis after five months.Reactivation of Bcr-Abl was confirmed by Crkl immunoblotting [K.Senechal, Mol. Cell. Biol. 18, 5082 (1998)]. The relative proportion ofphosphorylated Crkl (reflecting active Bcr-Abl) was 49% before STI571therapy, 24% at day 27, 28% at day 83, and 77% at the time of clinicalresistance at day 166. The biological significance of the Glu255Valchange is determined by an Abl autophosphorylation assay. STI571inhibits wild-type Abl with an IC₅₀ of 0.025 μM. The mutation leads to avirtual insensitivity to STI571, with an IC₅₀ of >5 μM.

Example 4

The Bcr-Abl kinase domain from cells obtained from 12 CML and Ph+ acuteleukemia patients who relapsed while receiving STI571 was sequenced. Afunctional point-mutation in the kinase domain in one case wasidentified. This was a G→A change that results in a Glu→Lys substitutionat position 255 of Abl.

Example 5 Patients and Sample Preparation

Thirty bone marrow samples from 21 patients with Ph+ ALL who wereenrolled into consecutive “Phase II study to determine the safety andanti-leukemic effect of STI571 in adult patients with Ph+ acuteleukemias” were analyzed. According to the study protocol, thesepatients had relapsed ALL or were refractory after at least 2 cycles ofstandard chemotherapy. From all of the patients samples were obtainedbefore beginning STI571 treatment: 13 of these samples were fromindividuals who later were classified as good responders to STI571 (Nos.1-13, sensitive, S) including 12 patients with hematological completeremission (CR) and one patient with partial remission (PR) but completeperipheral hematological recovery (No. 1). Eight samples were collectedfrom individuals who later were found not to respond to STI571 (Nos.14-21, primarily resistant, R) including 6 patients without anyhematological response, one with cytoreduction in the bone marrow butpersistent peripheral leukemic cells (No. 20) and another with PR butincomplete peripheral hematological recovery (No. 16). Matched bonemarrow samples from 9 patients (Nos. 1-5 and Nos. 14-17) were alsoobtained while they were on treatment with STI571. Mononuclear cellswere separated by density gradient centrifugation through Ficoll-Hypaque(Biochrom, Berlin, Germany). Total RNA was extracted using the acidguanidium/phenol/chloroform method with minor modifications [Puissant C.and Houdebine L. M., Biotechniques 8, 148-149 (1990)]. Only samples withleukemic blast cell infiltration of more than 80% were included into theanalysis.

Reverse Transcription Polymerase Chain Reaction and Sequencing Analysis

One microgram of total RNA was used for reverse transcription (RT) bySuperscript II RT (Life Technologies, Grand Island, N.Y.) according tostandard protocols. Primers specific for the ATP binding site of ABLincluding the “loop” were designed using gene bank informationGI6382056: ATP-F 5′-GCG CM CM GCC CAC TGT CT-3′; ATP-R 5′-GCA CTC CCTCAG GTA GTC CA-3′ and LOOP-F 5′-TGG ACT ACC TGA GGG AGT GC-3′; LOOP-R5′-CGG TAG TCC TTC TCT AGC AGC-3′. Oligonucleotides were synthesized byLife Technologies. Polymerase chain reaction (PCR) was performed asdescribed previously [Hofmann W. K. et al., Leuk. Res. 25, 333-338(2001)] using an annealing temperature of 58° C. PCR-products wereseparated on a 2% agarose gel containing 0.3 mg/ml ethidium bromide andpurified using the QIAquick purification system (Qiagen, Valencia,Calif.) according to the manufacturers protocol. The purified DNA wasdirectly sequenced in both directions (sense and antisense) by the ABIPRISM dye terminator cycle sequencing reaction (Perkin-Elmer, Foster,Calif.).

Results:

Analysis of the sequence of the ATP binding site revealed a single pointmutation at nucleotide 1127 (GI6382056) changing a G to an A resultingin a substitution at codon 255 of Lys (mutant) for a Glu (wild-type).This mutation was found in 6 samples from patients after they weretreated with STI571 (Nos. 1, 2, 4, 5, 15, 16) but mutations were notfound in any other sample including the matched samples from thepatients before beginning treatment with STI571 (Table 3). The changewas verified by sequencing from both the sense and antisense directions.In addition, one sample (No. 17) from a patient with an aberrant cALLhad a single point mutation at nucleotide 1308 changing a C to Tresulting in a substitution at codon 315 of isoleucine (mutant) for athreonine (wild-type). This sample was unusual because the cells alsoexpressed CD33, a cell surface protein expressed on myeloid cells.

Our data strongly suggest that E255K developed during treatment withSTI571. Our analysis of matched samples found, that none of the samplesfrom untreated patients (including sensitive patients and those withprimary resistance) had this mutation. In contrast, six of 9 samples(67%) from these patients undergoing treatment with STI571 had thissubstitution at E255. The overall frequency of mutations in the ATPbinding site was 7 of 9 (78%) in our paired bone marrow samples frompatients undergoing therapy with STI571.

TABLE 3 Matched bone marrow samples: Development of mutations in theregion coding for the ATP binding site of ABL during treatment of Ph+ALL with STI571. ABS ABS status prior to status after treatment Responseto treatment No. Diagnosis with STI571 STI571 with STI571 1 Ph+ cALLWild type PR E255K 2 Ph+ cALL Wild type CR E255K 3 Ph+ cALL Wild type CRWild type 4 Ph+ cALL Wild type CR E255K 5 Ph+ cALL Wild type CR E255K 14Ph+ cALL Wild type no Wild type 15 Ph+ cALL Wild type no E255K 16 Ph+pre B-ALL Wild type PR E255K 17 Ph+ cALL, CD33+ Wild type no T315I ABS,ATP binding site; PR, partial remission; CR, complete remission; Ph+cALL, Philadelphia chromosome positive common ALL (CD10+).

1. An isolated polypeptide which comprises a mutated functional Ablkinase domain that is resistant to inhibition of its tyrosine kinaseactivity byN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamideor a salt thereof.
 2. An isolated polypeptide according to claim 1,wherein the mutated functional AbI kinase domain comprises the aminoacid sequence of the native human Abl kinase domain or an essentiallysimilar sequence thereof in which at least one amino acid is replaced byanother amino acid.
 3. An isolated polypeptide according to claim 2,wherein in the amino acid sequence of the native human Abl kinase domainor an essentially similar sequence thereof at least one amino acidselected from Leu248, Glu255, Lys271, Glu286, Met290, Thr315, Tyr320,Asn322, Glu373, His375 and Ala380 is replaced by another amino acid. 4.An isolated polypeptide according to claim 3, wherein in the amino acidsequence of the native human Abl kinase domain or an essentially similarsequence thereof at least one amino acid selected from Leu248, Glu255,Lys271, Glu286, Met290, Tyr320, Asn322, Glu373, His375 and Ala380 isreplaced by another amino acid.
 5. An isolated polypeptide according toclaim 4, wherein in the amino acid sequence of the native human Ablkinase domain or an essentially similar sequence thereof at least oneamino acid selected from Leu248, Lys271, Glu286, Met290, Tyr320, Asn322,Glu373, His375 and Ala380 is replaced by another amino acid.
 6. Anisolated polypeptide according to claim 3, wherein in the amino acidsequence of the native human Abl kinase domain or an essentially similarsequence thereof at least one amino acid selected from Glu255, Thr315and Ala380 is replaced by another amino acid.
 7. An isolated polypeptideaccording to claim 6, wherein in the amino acid sequence of the nativehuman Abl kinase domain or an essentially similar sequence thereof atleast one amino acid selected from Glu255 and Ala380 is replaced byanother amino acid.
 8. An isolated polypeptide according to claim 2,wherein in the amino acid sequence of the native human Abl kinase domainor an essentially similar sequence thereof a single amino acid isreplaced by another amino acid.
 9. An isolated polypeptide according toclaim 7, wherein in the amino acid sequence of the native human Ablkinase domain or an essentially similar sequence thereof. Glu255 isreplaced by another amino acid.
 10. An isolated polypeptide according toclaim 3, wherein the amino acid sequence of the native human Abl kinasedomain or an essentially similar sequence thereof contains at least oneamino acid mutation selected from Glu255Val, Glu255Lys, Thr315Val,Thr315Leu, Thr315Met, Thr315Gln, Thr315Phe and Ala38Thr.
 11. An isolatedpolypeptide according to claim 10, wherein the amino acid sequence ofthe native human Abl kinase domain or an essentially similar sequencethereof contains at least one amino acid mutation selected fromGlu255Val, Thr315Val, Thr315Leu, Thr315Met, Thr315Gln, Thr315Phe andAla380Thr.
 12. An isolated polypeptide according to claim 11, whereinthe amino acid sequence of the native human Abl kinase domain or anessentially similar sequence thereof contains at least one amino acidmutation selected from Thr315Leu, Thr315Met, Thr315Gln and Thr315Phe.13. An isolated polypeptide according to claim 10, wherein the aminoacid sequence of the native human Abl kinase domain or an essentiallysimilar sequence thereof contains a single amino acid mutation.
 14. Anisolated polypeptide according to claim 11, wherein the amino acidsequence of the native human Abl kinase domain or an essentially similarsequence thereof contains the amino acid mutation Glu255Val.
 15. Anisolated polypeptide according to claim 2, wherein the amino acidsequence of the native human Abl kinase domain consists of amino acids229-500 of SEQ ID NO:2.
 16. An isolated polypeptide according to claim2, which is a Bcr-Abl tyrosine kinase.
 17. Use of a polypeptideaccording to claim 2 to screen for compounds which inhibit the tyrosinekinase activity of said polypeptide.
 18. An isolated nucleic acidmolecule comprising a nucleotide sequence that encodes a polypeptideaccording to claim
 2. 19. Use of a nucleic acid molecule according toclaim 18 in the production of a polypeptide according to claim 2 for usein screening for compounds which inhibit the tyrosine kinase activity ofsaid polypeptide.
 20. A recombinant vector comprising a nucleic acidmolecule according to claim
 18. 21. A recombinant vector according toclaim 20, which is a recombinant expression vector.
 22. A host cellcomprising a recombinant vector according to claim 20.